Zinc and iron are the two most abundant transition metals in biological systems, however the molecular mechanism of zinc and non-transferrin bound iron transport into cells is not resolved. Fourteen human ZIP family members have been identified based on sequence similarity to iron (Irt in S. cerevisiae) and zinc (Zrt in A. thaliana transporters. These parent members of the ZIP protein family have differing cation selectivity and it is currently unclear what molecular determinants define cation selectivity for the ZIP family of proteins. Through analysis of the human (h) Zn2+ importer, ZIP4, this proposal will define the contribution of the transmembrane, N-terminal and cytosolic domains in the ion permeation pathway and cation selectivity of the ZIP family of proteins as well as elucidate why previously discovered mutations in zinc transporters lead to distinct disease states. The long-term goal of the application is to elucidate the structure and function of a eukaryotic ZIP protein which is directly implicated in multiple disease states and resolve how transition metal transporters are selective for first row transition metals which have similar ionic radii, charge ad coordination geometry. The aims of this proposal are: 1) Test the hypothesis that residues within the transmembrane domains define both the transition metal permeation pathway and selectivity of ZIP transporters, 2) Test the hypothesis that zinc coordination to the cytosolic domain induces a conformational change which regulates the velocity of biometal translocation, and 3) Test the hypothesis that AE causing mutations within the N- terminal domain as well as zinc deficiency triggered cleavage of the large hZIP4 N-terminal domain, regulates zinc uptake. To address aim one, an uptake assay following heterologous expression of hZIP4 mutants in X. laevis oocytes will be employed to elucidate the contribution of targeted transmembrane domains in the ion permeation pathway and cation selectivity of hZIP4. To obtain the goals of aim two, residues that coordinate Zn2+ in the cytosolic domain will be identified and changes in Zn2+ uptake upon disruption of cytosolic Zn2+ co- ordination for the full-length protein will be measured. Finally, the goals of aim three will be accomplished by examining zinc uptake for hZIP4 and mutant constructs, including truncated hZIP4, to directly measure the effects of alterations of the N-terminal domain in hZIP4 on Zn2+ uptake. As zinc is required for life, the results from this study will be significant as it will provide a detailed description of the contribution of the extracellular, transmembrane and intracellular domains to the cation permeation pathway of ZIP proteins as well as define the molecular determinants which contribute to differing cation specificity among the ZIP family of proteins. The use of a combination of approaches, including transport assays, structural biology and biophysical methods, to systemically elucidate the transition metal permeation pathway and the selectivity determinants of a protein which is directly implicated in several human diseases, is novel, timely and innovative.